%0 Journal Article %T 越桔ISSR-PCR反应体系建立与优化<br>Establishment and Optimization of an ISSR-PCR Reaction System for <i>Vaccinium</i> Linn. %A 谌月 %A 鲁艳 %A 李凌< %A br> %A CHEN Yue %A LU Yan %A LI Ling %J 西南大学学报(自然科学版) %D 2017 %R 10.13718/j.cnki.xdzk.2017.09.005 %X 以改良CTAB法提取越桔(<i>Vaccinium</i> Linn.)品种薄雾DNA为材料,采用正交设计L<sub>16</sub>(4<sup>5</sup>)表探讨了Tag酶;Mg<sup>2+</sup>;DNA;dntps及引物浓度5个主要因素对越桔ISSR-PCR扩增反应的影响,通过直观分析和方差分析结合,建立了越桔ISSR-PCR(20 μL)最佳反应体系,各组分的浓度为:Tag酶0.75 U,Mg<sup>2+</sup>1.875 mmol/L,DNA 0.8 mmol/L,dntp 0.1 mmol/L,引物0.2 μmol/L.在此基础上探讨了引物UBC835的最佳退火温度、循环次数和延伸时间,结果分别为54.2 ℃、35次、60 s.选用两个引物对21份越桔资源进行ISSR扩增,结果显示优化的体系稳定性较高.<br>Based on the genomic DNA extracted from <i>Vaccinium</i> Linn. cultivar Misty with a modified CTAB method, five factors influencing ISSR were optimized in an orthogonal-design experiment. Through intuitive analysis and variance analysis, an optimal ISSR-PCR reaction system was established for this fruit crop, namely 10×buffer 2 μL; 0.75 U Taq DNA polymerase; 1.875 mmol/L Mg<sup>2+</sup>; 80 ng template DNA; 0.1 mmol/L dNTPs; and 0.2 μmol/L primer. The optimal annealing temperature, number of cycles and extension time of primer UBC835 were investigated, and the results were 54.2 ℃, 35 cycles and 60s, respectively. Twenty-one <i>Vaccinium</i> accessions were used to test the stability of the optimized reaction system. The results indicated that the optimized reaction system was very stable %K 越桔 %K 简单序列重复区间(ISSR) %K 正交设计 %K 直观分析 %K 方差分析< %K br> %K < %K i> %K Vaccinium< %K /i> %K Linn %K inter simple sequence repeat(ISSR) %K orthogonal design %K intuitive analysis %K variance analysis %U http://xbgjxt.swu.edu.cn/jsuns/html/jsuns/2017/9/201709005.htm